STEM CELLS IN REFRACTORY ASHERMAN SYNDROME
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1 STEM CELLS IN REFRACTORY ASHERMAN SYNDROME Dr Neeta Singh, MD, FICOG, FCPS, FIMSA Professor Division of Reproductive Medicine Department of Obstetrics & Gynecology All India Institute Of Medical Sciences New Delhi, India
2 INTRODUCTION Asherman syndrome (AS) an infrequent and complex gynecological abnormality due to damage to endometrium causing partial or complete obliteration of uterine cavity and/or cervical canal Symptoms- menstrual abnormalities (hypomenorrhoea or amenorrhoea), infertility and RPL Prevalence- 2% to 22% in infertile women Causes: Invasive curettage most commonly of gravid uterus % Trauma to non gravid uterus (diagnostic curettage - 1.6%, abdominal myomectomy - 1.3%, IUCD insertion - 0.2%) Infection - Genital TB, schistosomiasis Genetic predisposition Yu D et al. FertilSteril2008
3 Treatment of AS Laparoscopic guided Hysteroscopic lysis of adhesions Placement of physical barriers in uterine cavity Estrogen supplementation to enhance endometrial proliferation Agents to increase vascular flow to endometrium (Vitamin E, L- arginine, sildenafil citrate) BUT RECURRENT ADHESIVE DISEASE DESPITE ABOVE TREATMENT Recurrence rate for IUAs - 3.1% to 23.5% among all cases, % in those with severe adhesions TsuiKH et al. Taiwanese J ObstetGynecol2014
4 Persistent thin endometriumchallenge to clinician Thin endometrium despite treatment in AS lower implantation and pregnancy rate Ongoing treatment for thin endometrium- Intra-uterine granulocyte colony-stimulating factor Extended estrogen support Human chorionic gonadotropin priming in the follicular phase Drugs that increase endometrial blood flow (Pentoxyfilline tocopherol, sildenafil and l-arginine) Promising treatment in poor endometrium- stem cell based therapies
5 Pathophysiology Endometrium high capacity for self renewal cycles in reproductive life regulated by hormones Comparable to other high cellular turnover tissues gut, epidermis, bone marrow Histologically endometrium has 2 layers Functional layer- responds to hormone, shed at menses Basal layer- regenerates mucosa Endometrium derived stem cells (EDRSCs) present in niche in basal layer highly regenerative self renewal AS Absence of functional endometrium leads to infertility
6 STEM CELLS Emerging role in regenerative medicine treatment of tissue injury and fibrosis Stem cells - undifferentiated cells - potential to be multiplied in undifferentiated form (self renewal) and to mature and differentiated cells Self renewal and multi lineage differentiation treatment of degenerative disease Potential of substituting damaged cells in the endometrium Multipotent and pluripotent stem cells beneficial paracrine effects - decrease cell death and provide growth/trophic support to host cells
7 STEM CELLS - TYPES Two types Embryonic stem cells (ESCs) origin from blastocyst Adult stem cells (ASCs) Both types can further differentiate into : Totipotent stem cells- totally undifferentiated, potency to produce whole embryonic germ layers (ectoderm, endoderm, and mesoderm) such as zygote Pluripotent stem cells - end of a spectrum of differentiation, potential to generate all three germ layers cells, but not extraembryonic cells such as ICM of the blastocyst Multipotent stem cells can create variable but confined number of cell types limited to same lineage such as mesenchymal stem cells Unipotent stem cells stay restricted to the specific tissue and give rise to just one cell type
8 Sources Stem cells Source Properties Bone marrow derievedstem cell (BMDSC) CD45 + Hematopoeiticprogenitor stem cell (HPS) Human endometrial side population (hesp) Bone marrow Bone marrow Endometrium Low quantity, differentiation with age Mesoderm in red bone marrow, umbilical cord blood, peripheral blood, cross bone marrow barrier Produce endometrial, endothelial, epithelial and stromal cells Autologous adult stem cell Bone marrow Compensatory cells for generative tract Mesenchymal stem cell (MSC) Adipose tissue Easy separation, plentiful proliferation, weakly immunogenic, large volume Endometrial mesenchymal stem cell (emsc) Human endometrial stem cell (hesc) Human umbilical cord mesenchymal stem cell Endometrium Embryonic Umbilical cord Multipotent, vastly proliferative, easily collected All three germ layers, limitless capacity Best clinical tool, easy accessing, faster self renewal, HLA class 1 weakly expressed Azizi R et al. Biomedicine and Pharmacotherapy. 2018
9 Mesenchymal stromal cells Best option for cell/gene therapy and regenerative medicine in various tissues such as uterus - easy separation from various tissues plentiful proliferation capacity in vitro without any change in their biological features injured tissues tropism weakly immunogenic secretion of anti-inflammatory molecules lowest hurt to normal cells/tissues
10 Endometrial Stem Cells (EnSCs) Prianishnikov (1978) for the first time introduced endometrial stem cells (EnSCs) Chan et al. isolated EnSCs from endometrial tissue in 2004 Gargett et al. (2009) identified the extensive use of EnSCs in therapeutic applications EnSCs source of Mesenchymal stem cells (MSCs) Epithelial stem cells (ESCs) Endometrial side population (ESP) Endometrial regenerative cell (ERC)
11 Endometrial Stem Cells (EnSCs) Properties Self-renewal, high proliferative capacity Colony-forming unit activity capacity to differentiate into several cell types of mesodermal, ectodermal and endodermal origins Stem cells existing in endometrium might originate from bone marrow Bone marrow stem cells (BMSCs) might have important role in endometrial rebuilding Obstacle in BMSCs use low quantity, differentiation potency with age, aggressive seclusion method
12 MECHANISM
13 PROCEDURE
14 PROCEDURE Stem cell isolation and identification by flow cytometry Intravenous or intrauterine (preferred) administration of stem cells Stem cells encapsulated in biomaterials such as hydrogels and scaffold to increase retention and survival New cells stick to ECM that provides suitable milieu for growth and development ECM binds growth factors and restricts dissemination of stem cells
15
16 Endometrial regeneration using autologous adult stem cells followed by IVF in severe AS- Case report Severe AS- curettage fb IUCD and cyclical hormonal therapy for 6 months failed Autologous stem cells isolated from bone marrow endometrial angiogenic stem cells separated Intrauterine instillation under USG guidance fb cyclical hormonal therapy Endometrial thickness reassessed- 8 mm good vascularity IVF-ET done- positive pregnancy Nagoriet al. J Hum ReprodSciences 2011
17 STUDIES- ANIMAL AND HUMAN Non uterine stem cells can regenerate endometrium CD 45 cells migrate to endometrium in pregnancy Mesenchymal origin can form endometrium Endometrial regeneration after xenografting in kidneyl Cells deposited in cavity following curettage can reproduce endometrium MSCs added to estrogen stimulate regeneration BMDSC functional role in regeneration
18 STUDIES- ANIMAL AND HUMAN Collagen scaffolds support uterine repair Role in menstrual reconstruction Immunomodulatory role by locating in endometrium Clonogenic SUSD2+eMSCs recognised in regenerated endometrium Reduce fibrosis and enhance glandular count Expression of ER and PR enhanced Increased congestion of mature vessel and duration of menses Increased endometrial thickness Immunomodulatory role
19 CD133+ stem cells Bone marrow derived stem cells express biomarkers CD133/VEGFR2 subpopulation of cells with endothelial progenitor capacity (EPCs) Mobilised to peripheral circulation neoangiogenesis, healing, repair Can differentiate into non hematopoietic cell lineages Used in clinical trials in chronic total occlusion and ischemia, MI, hepatic fibrosis, liver regeneration, bone regeneration - excellent safety profile CD133+ cells recently categorized as Advanced Therapy Medicinal Product (ATMP) with regenerative properties Santamaria X. Encyclopediaof Reproduction 2 nd Edition, Vol
20 MOA
21 Prospective, experimental, noncontrolled, open label study- 18 patients (30 45 yrs) with refractory AS or EA 16 completed study Recombinant human granulocyte colony stimulating factor (G-CSF) -10 mg/kg/day X 5 day Peripheral Blood Mononuclear Cells isolated by apheresis - CD133+ cells isolated Cells delivered into spiral arterioles by catheterisation Significant improvement in duration and intensity of menstrual period in first 3 months Statistically significant improvement in endometrial thickness Reproductive outcome- 3 patients spontaneous pregnancy, 7 pregnancy after ET
22 OUR EXPERIENCE
23 Prospective case series 6 cases of refractory AS with failed standard treatment Resident endometrial stem or progenitor cells can be activated and bone marrow derived stem cells can be transplanted into uterine cavity for regeneration Mononuclear stem cells (MNCs) implanted in subendometrial zone followed by oral estrogen for 12 weeks ET was assessed at 3, 6, and 9 months
24 Contd Procedure Bone Marrow (30 ml) aspirated from iliac crest with BM aspiration needle (Jamshidi, 11 G) in heparinized syringe Mononuclear cells (MNCs) isolation done by Ficoll density separation method MNCs suspended in 3 ml heparinized NS evaluated for viability (trypan blue dye), morphology and CD 34+ cells 3 ml of MNC injected in subendometrial zone at 2-3 sites (fundus, anterior and posterior part) under TVS guidance with ovum aspiration needle (17 gauge) Singh N et al, J Hum Reprod Sci.2014 Apr-Jun; 7(2): 93 98
25 Singh N et al, J Hum Reprod Sci.2014 Apr-Jun; 7(2): 93 98
26 Contd.. CD34+- endothelial progenitor cells that promote angiogenesis and tissue repair The correlation between no of MNC cells or CD 34+ cells and increase in ET was not significant. Singh N et al, J Hum Reprod Sci.2014 Apr-Jun; 7(2): 93 98
27 Contd.. The maximum ET achieved by any patient was 6.7 mm 1st study where adult stem transplant tried in patients of Asherman s due to genital TB CONCLUSION Autologous stem cell implantation- innovative therapy for endometrial regeneration in AS. Since women resumed menstruation after implantation this therapy holds promise for future fertility in these patients Singh N et al, J Hum Reprod Sci.2014 Apr-Jun; 7(2): 93 98
28 Autologous bone marrow derived stem cell therapy for Asherman s syndrome and endometrial atrophy: 5 year follow up study Prospective study - 25 women (24-37 years) with Asherman s syndrome or endometrial atrophy after failed standard treatment Study duration: November 2011 and January 2013 MNCs in 5 ml heparinized NS injected in uterine cavity through 6F pediatric foleys catheter and kept in situ for 36 hrs. Post procedure oral estradiol valerate tablets 2 mg thrice daily for 12 weeks ET and menstrual pattern assessed at 3, 6, and 9 months and at 5 years UNPUBLISHED DATA. Singh N
29 Baseline characteristics Baseline characteristics Mean + SD/Percentage Age (years) BMI (kg/m 2 ) FSH (IU/ml) LH (IU/ml) Contd.. Genital TB (%) 60 Change in ET at 9 months follow up Pre-ET ET at 3 months P value ET at 6 months P value ET at 9 months P value UNPUBLISHED DATA. Singh N
30 Contd.. Post stem cell treatment AT 3 months (ET-6.6 mm) Pre stem cell treatment endometrial thickness of a patient (ET 3.3 mm) AT 6 months (ET-6.8 mm)
31 Asherman syndrome No Age Cause 1 29 C sec Preop menstrual history Amenorrhea 1.2 Max preop ET Hysteroscopy Preop AS IV Postop (5yrs) AS II Postop menstrual history Max postop ET Scanty 6.7 (6 months) Postop 4D USG Tubular cavity Pregnancy outcome No 2 34 D&C Amenorrhea 2.2 AS III - Normal 3 30 TB Amenorrhea 1.7 AS III AS II Scanty 4 23 TB Scanty 3.3 AS I Normal Normal 5 30 TB Scanty 3.8 AS I Normal Normal 6 25 TB Amenorrhea 2.8 AS III AS III Scanty 6.8 (9 Spontaneous months) conception, 38 wks, VD, 2750 gm 6.4 (6 months) 7.4 (5 years) Tubular cavity Normal No 6.9 (6 Normal months) 4.4(5 years) Tubular cavity No No No
32 No Age Cause Asherman syndrome Preop menstrual history Max preop ET Hysteroscopy Postop menstrual history Preop Post op 7 24 D&C Amenorrhea 2.1 AS II Scanty 5 (9 mo) 8 32 TB Scanty 4.6 AS I - 6 mo- N, scanty at 9 mo 9 29 TB Scanty 3.7 AS I 3 months- N scanty at 6 and 9 mo Max postop ET 8.3 (9 mo) 7.1 (6 mo) TB Scanty 3.2 AS II Scanty 5.9 (9 mo) TB Amenorrhea 2 AS II Amenorrhea 6.4 (6 mo) Pregnancy outcome Spont, 35 wks- CS, 2250 gm No No No No TB Amenorrhea 3 AS IV Normal at 3 mo, scanty at 6, 9 mo 6.2 (9 mo) No
33 Endometrial atrophy No Age Cause 1 27 TB Preop menstrual history Max preop ET Scanty 3.6 Pale Hysteroscopy Preop Postop (5yrs) Postop menstrual history Max postop ET Scanty 6.1 (9 months) Pregnancy outcome 2 yrs, ectopic, salpingectomy 2 30 D&C Scanty 3.4 Pale scanty till 6 months then normal yrs,ivf (9 months) conception, 34 wks, CS, 1500 gm 3 28 unknown Scanty 3.3 Pale Normal Normal 4 35 D&C Scanty 3.5 Pale Normal Normal 5 25 TB Scanty 4.1 Pale Normal Normal 6 24 unknown Scanty 4.9 Pale Pale normal till 3 months then scanty 6.6 (9 months) No 7 (5 years) No 9.3 (5 years) No 8.8 (9 months) No
34 Endometrial atrophy No Age Cause Preop menstrual history Max preop ET Hysteroscopy Preop Postop (5yrs) Postop menstrual history 7 33 TB Scanty 3.5 Normal Normal scanty till 3 months then normal Max postop ET 12 (3 months) Pregnan cy outcome no 8 38 TB Normal 2.5 Normal normal Normal 8.2 No (5 years) 9 30 CS Normal 4.2 Normal Normal Normal 6.4 No (5 years) unknown Normal 4 Pale Normal 6.3 (6 months) CS Normal 4 Pale Normal 9.1 (6 months) No TB Normal 4.5 Normal Normal 7.5 (9 months) TB Scanty 4.7 Pale normal till 3 months 5.6 then scanty (3 months) No No No
35 RESULTS RESULTS Majority of patients with amenorrhea resumed menses post stem cell instillation (6 out of 7) Duration and intensity of menses - 20 patients with scanty or no menses progressively increased 3 and 6 months post therapy but declined at 9 months ET significantly increased after 3 months of therapy following which it plateaued 4 pregnancies achieved in 5 year follow up period. 2 conceived spontaneously and two through ART. 3 had a successful reproductive outcome and 1 was an ectopic pregnancy UNPUBLISHED DATA. Singh N
36 CONCLUSION Regenerative medicine may play an important future role in the treatment of incurable infertility in AS Further studies are mandatory to better other important issues such as dosage, MoA and refining the protocol CD133+ cells as the first orphan drug designed (ODD) for the treatment of AS
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